The long-term goal of this laboratory is to understand the process of ubiquitin conjugation in the regulation of immune responses. Protein ubiquitination is carried out by a cascade of enzymatic reactions. In this process, the C-terminal glycine residue of the ubiquitin is catalyzed to form an isopeptide bond with the lysine residue of either a protein substrate or another ubiquitin molecule. Ubiquitination is a reversible process, which is driven by deubiquitinating enzymes that cleave the isopeptide bond between the ubiquitin and the substrate. A genomic bioinformatics study suggests that there are about 100 deubiquitinating enzymes in the human genome. In addition, bacterial and viral deubiquitination enzymes have also been discovered and are involved in pathogenesis. Compared with the process of protein ubiquitin conjugation, our knowledge on the mechanisms of ubiquitin removal or deubiquitination is far less. We initiated this project by screening the gene expression profile of deubiquitinating enzymes in purified mouse CD4+ T cells and selected a few of previously uncharacterized enzymes for further biochemical and functional analysis. We have obtained preliminary results suggesting that some of the enzymes are involved in T cell signaling. Based on these preliminary studies, we hypothesize that protein deubiquitination plays an important role in regulating T cell activation via catalyzing the deubiquitination of its target proteins. In this grant application, we will propose both biochemical and genetic approaches to test this hypothesis. The proposed studies will allow us to obtain important information on protein deubiquitination in the regulation of T cell activation and T cell-mediated immune response. Such knowledge will shed light on the design of new therapeutic approaches for immunological diseases. Protein deubiquitination in lymphocyte signaling Narrative Ubiquitin is a small molecule that signals for destruction when attached to a protein. The ubiquitin tagging is reversible process that is carried out by deubiquitinating enzymes. The proposal will aim at the understanding of the mechanisms of ubiquitin removal, particularly its implication in lymphocyte function. The knowledge gained from the proposed research will help design new therapeutic approaches for immunological diseases.